The mechanism of action and regulation of the alpha-ketoglutarate dioxygenase reaction will be investigated through the study of thymine 7-hydroxylase, pyrimidine deoxyribonucleoside 2'-hydroxylase and prolyl hydroxylase. Streptomyces antibioticus and the simple eukaryotes Neurospora crassa and Rhodotorula glutinis will serve as sources for enzyme purification. Among the techniques used to study the purified enzymes will be those for detecting and isolating oxygenated reaction intermediates. The intact cells of the above mentioned organisms, as well as of mammals, will also be studied, especially in regard to the factors which regulate the activities of the alpha-ketoglutarate dioxygenases. The objective of this proposal extends to the elucidation of the regulation and physiological functions of the oxidative and reductive pathways of pyrimidine catabolism and to gaining some insight into whether prolyl hydroxylase itself has a regulatory role.